The synthesis method of oligonucleotide includes a phosphate triester method, an H-phosphonate method, a phosphoramidite method and the like, and solid phase synthesis (solid phase method) using a phosphoramidite method is most widely used at present (non-patent document 1). The solid phase method is advantageous from the aspect of speed, since process has been optimized and automation has progressed. However, it is associated with defects in that scaling-up is limited due to facility restriction, reagents and starting materials are used in excess, and confirmation of the progress status of the reaction in an intermediate step, analysis of intermediate structure and the like are difficult.
The synthesis methods of oligonucleotide by a liquid phase method have also been studied. Generally, however, treatments after each reaction are performed by a method including (1) directly concentrating the reaction mixture, followed by isolation and purification by silica gel column chromatography, (2) extracting with a solvent such as methylene chloride, chloroform and the like, washing with an aqueous solution, concentrating, purifying by silica gel column chromatography, and the like, and the operation is complicated and the yield is low. In particular, a large-scale, rapid synthesis of a long oligonucleotide is difficult, and the methods are impractical as industrial production processes.
In recent years, a pseudo-solid phase method-like approach has been reported as an attempt to solve the respective defects of the liquid phase method and the solid phase method, and an oligonucleotide production method using a soluble polymer such as monomethoxypolyethylene glycol (MPEG) and the like as a protecting group is disclosed as one example thereof (non-patent document 2). However, while synthetic examples of up to 20 mer DNA are disclosed, a crystallization isolation operation is essential for each reaction, and the progress status of the reaction and the like are difficult to confirm, since MPEG molecule itself is not a unimolecule.
In addition, as a pseudo-solid phase method-like method, a production method of oligonucleotide including use of an ionic liquid as a protecting group has been reported, and Synthetic Examples of DNA up to pentamers are shown (non-patent document 3). However, the method is inferior to a solid phase method in the speed and efficiency, since a crystallization isolation operation is essential for each reaction and an operation time is necessary therefor.
Furthermore, a synthesis method of oligonucleotide comprising use of a hydrophobic group-linked nucleoside is disclosed (patent document 1). While it has been reported that the method affords synthesis of 21 mer oligonucleotide, it is markedly complicated, since the method requires solidification isolation in every step of deprotection of 5′-protecting group, coupling and oxidation.